Figure 1.

(a) Schematic of the two-photon lifetime microscopy system. Excitation light is provided by a laser oscillator (MaiTai-BB) through an acousto-optic modulator (AOM) followed by a polarizer (P) to adjust the gain. A telescope (L₁ and L₂) expands the galvo-mirrors image onto the microscope objective pupil for illumination. Emitted fluorescence is separated using a first dichroic mirror (DM₁). The return beams are then split by a second dichroic mirror (DM₂) sending the signal to detectors centered at wavelengths of 520 nm (F₁) and 593 nm (F₂). The Ca²⁺ concentration was monitored in the 520 nm channel using a photon-counter for FLIM imaging. (b) Top: In vivo fluorescence staining of neurons in green, astrocytes in yellow and vasculature in red. Bottom: OGB-1 stained neurons and astrocytes (left panel) while SR101 stained astrocytes alone and Rhodamine B localized the vasculature (right panel). (c) Pictogram of measurement areas on the mouse brain. The craniotomy was done on the left side. The 4-AP injection location and electrode recordings were done at the same site. The remote area was defined to be that further than 1.5 mm from the focus. (d) Calibration of the fluorescence decay of OGB-1 at 10 different buffer Ca²⁺ concentrations. In this range, fit lifetime varied from ∼4.86 ns to ∼0.65 ns for high/low concentrations, respectively. (e) Typical images at 593 nm for longitudinal vessel scan used to measure the diameter. The right figure shows measurements of absolute [Ca²⁺]_i in one astrocytic endfoot during resting state.